3-TYP

SIRT3 ( IC50 = 16 nM ); SIRT1 ( IC50 = 88 nM ); SIRT2 ( IC50 = 92 nM )

3-TYP 抑制褪黑激素增强的 SIRT3 活性，但不影响 SIRT3 蛋白表达。 3-TYP 预处理可逆转褪黑激素对镉 (Cd) 诱导的线粒体 O2•− 产生和自噬细胞死亡的保护作用。 3-TYP 显着减弱褪黑激素诱导的暴露于 Cd 的 HepG2 细胞中脱乙酰 SOD2 表达和 SOD2 活性的增加[1]。

与 Sham 组相比，3-TYP（50 mg/kg，ip）对 LVEF、LVFS、梗塞面积、血清 LDH 水平、细胞凋亡和氧化应激没有显着影响。此外，与Sham组相比，3-TYP对gp91phox、Nrf2、NQO 1、Bax、Bcl-2、Caspase-3和cleaved Caspase-3表达水平几乎没有影响。与对照组相比，3-TYP显着降低SIRT3活性并增加SOD2乙酰化，但不影响SIRT3表达。 3-TYP 通过降低再灌注 24 小时后的 LVEF 和 LVFS 来减弱褪黑激素的心脏保护作用。与 IR+Mel 组相比，3-TYP 还增加了梗死面积、血清 LDH 水平和细胞凋亡率 [2]。

SIRT3抑制剂（3-TYP）用于证实褪黑素参与镉诱导的自噬，并破坏SIRT3调节的线粒体来源的O2•−产生。3-TYP是一种选择性SIRT3抑制剂。31暴露于3-TYP会抑制褪黑素增强SIRT3活性，但不影响SIRT3蛋白表达（图S6A和B）。此外，3-TYP预处理逆转了褪黑素对镉诱导的线粒体来源的O2•−产生和自噬细胞死亡的保护作用（图9A-C和图S3F）。如图9D和9E所示，在暴露于镉的HepG2细胞中，3-TYP显著减弱了褪黑素诱导的脱乙酰基-SOD2表达和SOD2活性的增加[1]。

使用 Cell Counting Kit-8 分析细胞活力。简而言之，将1×104个细胞接种到96孔板中。处理后，每孔加入90μL培养基和10μL CCK-8溶液。然后将细胞在 37°C 下孵育 2 小时。孵育后，使用 Infinite™ M200 酶标仪测量 450 nm 处的吸光度。结果以对照的百分比表示。还使用台盼蓝测定法评估细胞死亡。 HepG2细胞接种于6孔板中（每孔5×105个细胞）并孵育24小时。用 Cd 或褪黑激素处理后，用 300 μL 胰蛋白酶-EDTA 溶液分离细胞。将分离的细胞混合物以 300 g 离心 5 分钟。然后，将残余物与800μL台盼蓝溶液混合并分散。染色 3 分钟后，使用自动细胞计数器对细胞进行计数。死亡细胞被染成蓝色。细胞死亡率(%)表示为死细胞数/总细胞数的百分比。

In brief, a 6-0 silk suture slipknot is wrapped around the left anterior descending coronary artery to temporarily exteriorize the heart in male C57BL/6 mice under 2% isoflurane anesthesia. Following 30 minutes of myocardial ischemia, the myocardium is reperfused for 3 hours (to measure oxidative stress and perform a western blot analysis) or 24 hours (to assess infarct size, cardiac function, and apoptotic index). The slipknot is then released. The identical surgical procedures are performed on sham-operated mice, with the exception that the suture under the left coronary artery is left untied. Mice are randomized to receive an intraperitoneal injection of either melatonin (20 mg/kg) or vehicle (1% ethanol) ten minutes prior to reperfusion. The C57BL/6 mice are split into the following groups at random: (i) Sham group: mice underwent the sham operation and are treated with vehicle (1% ethanol); (ii) Mel group: mice are treated with melatonin (20 mg/kg via intraperitoneal injection); (iii) IR+V group: mice underwent the MI/R operation and are treated with vehicle (1% ethanol); (iv) IR+Mel group: mice underwent the MI/R operation and are treated with melatonin (20 mg/kg via intraperitoneal injection 10 minutes before reperfusion); (v) IR+Mel+3-TYP group: mice are pretreated with 3-TYP (3-TYP is intraperitoneally injected at a dose of 50 mg/kg every 2 days for a total of three doses prior to the MI/R surgery), subjected to the MI/R operation, and treated with melatonin (20 mg/kg via intraperitoneal injection 10 minutes before reperfusion); and (vi) IR+3-TYP group: mice are pretreated with 3-TYP and then subjected to the MI/R operation.

[1]. SIRT3-SOD2-mROS-dependent autophagy in cadmium-induced hepatotoxicity and salvage by melatonin. Autophagy. 2015;11(7):1037-51.

[2]. Melatonin ameliorates myocardial ischemia reperfusion injury through SIRT3-dependent regulation of oxidative stress and apoptosis. J Pineal Res. 2017 Sep;63(2).

[3]. Identification of a sirtuin 3 inhibitor that displays selectivity over sirtuin 1 and 2. Eur J Med Chem. 2012 Sep;55:58-66.

Cadmium is one of the most toxic metal compounds found in the environment. It is well established that Cd induces hepatotoxicity in humans and multiple animal models. Melatonin, a major secretory product of the pineal gland, has been reported to protect against Cd-induced hepatotoxicity. However, the mechanism behind this protection remains to be elucidated. We exposed HepG2 cells to different concentrations of cadmium chloride (2.5, 5, and 10 μM) for 12 h. We found that Cd induced mitochondrial-derived superoxide anion-dependent autophagic cell death. Specifically, Cd decreased SIRT3 protein expression and activity and promoted the acetylation of SOD2, superoxide dismutase 2, mitochondrial, thus decreasing its activity, a key enzyme involved in mitochondrial ROS production, although Cd did not disrupt the interaction between SIRT3 and SOD2. These effects were ameliorated by overexpression of SIRT3. However, a catalytic mutant of SIRT3 (SIRT3(H248Y)) lacking deacetylase activity lost the capacity to suppress Cd-induced autophagy. Notably, melatonin treatment enhanced the activity but not the expression of SIRT3, decreased the acetylation of SOD2, inhibited mitochondrial-derived O2(•-) production and suppressed the autophagy induced by 10 μM Cd. Moreover, 3-(1H-1,2,3-triazol-4-yl)pyridine, a confirmed selective SIRT3 inhibitor, blocked the melatonin-mediated suppression of autophagy by inhibiting SIRT3-SOD2 signaling. Importantly, melatonin suppressed Cd-induced autophagic cell death by enhancing SIRT3 activity in vivo. These results suggest that melatonin exerts a hepatoprotective effect on mitochondrial-derived O2(•-)-stimulated autophagic cell death that is dependent on the SIRT3/SOD2 pathway.[1]

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Sirtuins are a family of highly evolutionarily conserved nicotinamide adenine nucleotide-dependent histone deacetylases. Sirtuin-3 (SIRT3) is a member of the sirtuin family that is localized primarily to the mitochondria and protects against oxidative stress-related diseases, including myocardial ischemia/reperfusion (MI/R) injury. Melatonin has a favorable effect in ameliorating MI/R injury. We hypothesized that melatonin protects against MI/R injury by activating the SIRT3 signaling pathway. In this study, mice were pretreated with or without a selective SIRT3 inhibitor and then subjected to MI/R operation. Melatonin was administered intraperitoneally (20 mg/kg) 10 minutes before reperfusion. Melatonin treatment improved postischemic cardiac contractile function, decreased infarct size, diminished lactate dehydrogenase release, reduced the apoptotic index, and ameliorated oxidative damage. Notably, MI/R induced a significant decrease in myocardial SIRT3 expression and activity, whereas the melatonin treatment upregulated SIRT3 expression and activity, and thus decreased the acetylation of superoxide dismutase 2 (SOD2). In addition, melatonin increased Bcl-2 expression and decreased Bax, Caspase-3, and cleaved Caspase-3 levels in response to MI/R. However, the cardioprotective effects of melatonin were largely abolished by the selective SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl)pyridine (3-TYP), suggesting that SIRT3 plays an essential role in mediating the cardioprotective effects of melatonin. In vitro studies confirmed that melatonin also protected H9c2 cells against simulated ischemia/reperfusion injury (SIR) by attenuating oxidative stress and apoptosis, while SIRT3-targeted siRNA diminished these effects. Taken together, our results demonstrate for the first time that melatonin treatment ameliorates MI/R injury by reducing oxidative stress and apoptosis via activating the SIRT3 signaling pathway.[2]

C7H6N4

146.1493

146.059

C, 57.53; H, 4.14; N, 38.34

120241-79-4

9833992

White to off-white solid powder

0.866

54.46

1

3

1

11

128

0

N1=C(C([H])=NN1[H])C1=C([H])N=C([H])C([H])=C1[H]

VYXFEFOIYPNBFK-UHFFFAOYSA-N

InChI=1S/C7H6N4/c1-2-6(4-8-3-1)7-5-9-11-10-7/h1-5H,(H,9,10,11)

3-(2H-triazol-4-yl)pyridine

3 TYP; 3-TYP; 120241-79-4; 3-TYP; 3-(1H-1,2,3-triazol-4-yl)pyridine; Pyridine(3-TYP); 3-(1H-1,2,3-triazol-4-yl) pyridine; CHEMBL373134; MFCD25956467; Pyridine, 3-(1H-1,2,3-triazol-5-yl)-; 3TYP

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 29~125 mg/mL (198.4~855.3 mM)
Water : ~1.3 mg/mL (~8.6 mM)
Ethanol : 16.7~29 mg/mL (114.1~198.4 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (14.23 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (14.23 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (14.23 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。